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Human gastric cancer cell line; RNA-seq analysis of SNU-16 clones cells

ABSTRACT: Chromosomal instability in gastric cancer cells is associated with the amplification of oncogenes that encode receptor tyrosine kinases (RTKs), such as HER2 and FGFR2; such gene amplification varies from cell to cell and manifests as genetic heterogeneity within tumours. The intratumoural genetic heterogeneity of RTK gene amplification causes heterogeneity in RTK protein expression, which has been suggested to be associated with therapeutic resistance to RTK inhibitors; however, the underlying mechanism is not fully understood. Here, we show that extrachromosomal DNA (ecDNA) causes intratumoural genetic heterogeneity in RTKs and drug resistance due to diverse dynamic changes. We analysed the dynamics of FGFR2 and MYC ecDNA in a gastric cancer cell line after single-cell cloning. Similar to those in parental cells, the copy numbers of FGFR2 and MYC in subclones differed significantly between cells, indicating intraclonal genetic heterogeneity. Furthermore, the ecDNA composition differed between subclones, which affected FGFR2 protein expression and drug sensitivity. Interestingly, clone cells that were resistant to the FGFR2 inhibitor AZD4547 presented diverse changes in ecDNA, including chimeric ecDNA, large ecDNA and increased ecDNA numbers; these changes were associated with high expression and rephosphorylation of FGFR2. Conversely, when resistant clone cells were cultured under conditions that excluded AZD4547, the ecDNA status became similar to that of the original clone cells, and the inhibitory effect on cell growth was restored. Our results show that dynamic quantitative and qualitative changes in ecDNA can drive the intratumoural genetic heterogeneity of RTKs and resistance to RTK inhibitors.

ORGANISM(S): Homo sapiens

PROVIDER: GSE285828 | GEO | 2025/01/31

REPOSITORIES: GEO

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Project description:Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. EcDNA renders tumours treatment-resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA (ssDNA), leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds ssDNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer. This work was delivered as part of the eDyNAmiC team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK (CGCATF-2021/100012 and CGCATF-2021/100025) and the National Cancer Institute (OT2CA278688 and OT2CA278635) to H.Y.C., P.S.M., and V.B. This project was supported by NIH RM1-HG007735 (H.Y.C., W.J.G., C.C.).

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Human gastric cancer cell line; RNA-seq analysis of SNU-16 clones cells

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